Tissue engineering (TE) is poised to make an enormous impact on the range of therapeutic options for treatment of life-threatening conditions that afflict millions of Americans. To date, however, no marketed TE product has proven to be commercially successful. One of the primary factors contributing to this lack of profitability is the need for aseptic processing at all steps. Unlike traditional pharmaceutical and biotechnology products, engineered tissues and cell-based therapies must be processed aseptically throughout their manufacture. For this reason, these therapies come with extremely high facility and production costs. New, innovative approaches for aseptic processing and scale-up are required before cell- based therapies can become accessible to patients in the US health care system. Stratatech Corporation has approval from the FDA to begin Phase I/I I clinical trials with StrataGraft(tm), a human skin substitute tissue based on Stratatech's patented NIKS(tm) human keratinocyte cell line. In addition, Stratatech is developing a pipeline of genetically enhanced living skin products (ExpressGraft(tm)) for use in the chronic, non-healing diabetic, venous stasis, and decubitus skin wound markets. This Fast Track proposal is designed to develop a closed, aseptic manufacturing process for these innovative products which will greatly reduce production costs and increase patient safety. In Phase I we will address the issue of closed, aseptic keratinocyte culture and scale-up as well as the PHS and FDA concerns regarding xenotransplantation. We will determine parameters for cryopreservation of NIKS(tm) keratinocytes in sterile bag format. We will then thaw and propagate these preserved NIKS(tm) cells in closed cell factories without the aid of murine feeder cells. Finally, we will scale-up methods for NIKS(tm) culture to enable harvest and passage at a sufficient scale for full scale commercial manufacture. In Phase II we will extend these results by developing methods for production of StrataGraft(tm) skin tissue in closed tissue growth chambers. We will construct prototype chambers and test their ability to produce skin tissue in batch and perfusion modes with on-line process monitoring. Then, we will design an optimized second-generation tissue growth chamber which would also function as the final storage container. RELEVANCE: This proposal will develop innovative closed, aseptic processing methods for the manufacture of engineered living human skin substitute products, StrataGraft(tm) and ExpressGraft(tm) skin tissue, in development at Stratatech Corporation. By manufacturing these products in a closed, aseptic fashion without the need for mouse feeder cells, we will bring these breakthrough therapies to millions of Americans afflicted with chronic, non-healing diabetic, venous stasis, and pressure ulcers. [unreadable] [unreadable]